PROJECT SUMMARY/ABSTRACT Neurofilaments are space-filling cytoskeletal polymers that function to increase the cross-sectional area of axons. These polymers are transported along axons at fast rates, but the overall rate is slow because the movements are interrupted by prolonged pauses. Neurofilaments accumulate in axons during the growth of axonal caliber and they also accumulate abnormally and excessively in a wide range of neurodegenerative diseases, most notably amyotrophic lateral sclerosis. Mutations that disrupt neurofilament assembly also cause one form of Charcot-Marie-Tooth disease. The long-term goal of our research is to understand the transport and assembly dynamics of neurofilaments in axons and the mechanisms that cause neurofilaments to accumulate in development and disease. We propose three specific aims. Aim 1 is to investigate the assembly dynamics of neurofilaments in axons. Axonal neurofilaments can be 100 m or more in length in vivo and they can also exchange subunits with a soluble pool, but little is known about how these processes occur. We propose that neurofilaments can lengthen by end-to-end annealing of pre-formed filaments and that they can exchange subunits by addition and loss of subunits along the length of the filaments, a process that we term intercalary subunit exchange. We will use cell fusion as well as photobleaching and photoactivation strategies to test these hypotheses in cultured neurons. We will also extend our studies to other types of intermediate filament proteins to establish whether our findings are more generally applicable. Aim 2 is to investigate the role of phosphorylation in neurofilament transport. We propose that phosphorylation of neurofilaments by CDK5 and ERK1/2 kinases regulates their transport in an additive and dose-dependent manner by increasing the proportion of the time the neurofilaments spend pausing. We will test these hypotheses in cultured neurons by using site-directed mutagenesis to mimic phosphorylated or non- phosphorylated states at specific sites. We will also manipulate kinase activities directly using pharmacological inhibitors and constitutively active or dominant negative kinase constructs. Aim 3 is to investigate the functional significance of neurofilament pausing. We propose that neurofilament pausing is a critical determinant of axonal neurofilament content. We will test the hypothesis that neurofilament phosphorylation causes axonal neurofilaments to accumulate by increasing their residence time in axons. We will also use long-term myelinating cultures to test the hypothesis that myelinating cells locally slow neurofilament transport by increasing the proportion of the time that they spend pausing. The use of live-cell imaging to study neurofilament transport in myelinating axons in culture is a particularly innovative aspect of this Aim. The mechanisms that regulate neurofilament pausing are likely targets for disease processes that lead to excessive accumulation of neurofilaments in axons.